The diagnosis of infectious disease has traditionally relied upon microbiological culture methods to identify the causative organism and determine the appropriate antimicrobial treatment. This has remained so despite recent advances in molecular and immunological diagnostics. While the development of rapid and automated methods has served to increase the efficiency of microbiological analysis, traditional quantitative culture methods remain critical for definitive diagnosis of urinary tract and other infections (Baron & Finegold, Diagnostic Microbiology, 8th ed., C. V. Mosby, [1990], p. 253).
After proper specimen collection and transport, the laboratory professional must determine which of a multitude of culture media are most appropriate to use with the culture at hand. It is important to consider the type of specimen (e.g., urine, blood, sputum, etc.), and the most commonly isolated organisms associated with disease or infection at the site of specimen collection. The time and cost necessary to achieve a final diagnosis also must be borne in mind.
With respect to the type of specimen, there are considerations related to the normal flora from which the pathogens must be differentiated. This is particularly true for fecal, rectal, vaginal, buccal and other samples which commonly contain a characteristic background flora. Urine, a fluid which is normally sterile when excreted from the kidneys, often becomes contaminated with flora from the urethra, urethral opening and skin. Indeed, as voided, urine is by no means sterile. The first voided 10 ml volume of urine can contain up to 10.sup.4 organisms per ml, due to the dislodgement of bacteria from the urethra This necessitates the differentiation of normal flora contaminants from the infecting organism(s).
As with most body sites, the normal urethra supports a characteristic normal flora. In females, the organisms comprising the normal flora vary with age and health. In premenarchal females, 66% of the organisms are aerobic coryneforms, lactobacilli, and coagulase-negative staphylococci. Streptococci are often present also. In women of reproductive age, lactobacilli are the most common isolates (Clarridge et al., "Laboratory Diagnosis of Urinary Tract Infections," Cumitech 2A, p. 1, American Society for Microbiology, 1987). In post-menopausal women, there is a marked increase in the number of anaerobes, particularly Bacteroides melaninogenicus (Clarridge et al., p. 1 ). Other organisms, such as mycoplasmas and low densities of enteric gram-negative rods may also be recovered from the urethra of healthy women (Clarridge et al.).
In males, less indigenous flora is isolated from urine. Coagulase-negative staphylococci, enterococci (i.e., group D streptococci), coryneforms, and mycoplasmas may be isolated from the urethra and urine of healthy men (Clarridge et al., supra). Table 1, lists the commensal flora (i.e., normal flora) associated with the human urinary tract.
TABLE 1 ______________________________________ Commensal Flora Associated With The Urinary Tract* Resident Flora of the Urethra ______________________________________ Coagulase-negative Staphylococci Viridans and Non-Hemolytic Streptococci Lactobaccilli Corynebacterium sp. (diphtheroids) Neisseria (non-pathogenic species) Transient Gram-Negative Aerobes (including Enterobacteriaceae) Anaerobic Cocci Propionibacterium sp. Anaerobic Gram-Negative Cocci and Bacilli Commensal Mycobacterium sp. Commensal Mycoplasma sp. Occasional Yeasts ______________________________________ *Koneman et al., Color Atlas and Textbook of Diagnostic Microbiology, 4th edition, (p. 79) (J. B. Lippincott Co., 1992); Baron & Finegold, Diagnostic Microbiology, 8th ed., pp. 253-262 (C. V. Mosby, 1990); and Power & McCuen, Manual of BBL .RTM. Products and Laboratory Procedures, 6th ed., pp. 48-49 (Becton Dickinson Microbiology Systems, 1988).
Because of the associated normal flora and the desire to identify pathogenic organisms, methods of urine collection have been developed which minimize the chances of contamination, including the clean-catch midstream sample, careful catheterization, suprapubic aspiration, bladder washout, and cystoscopy. In situations where the patient cannot or will not provide a clean-catch sample, suprapubic aspiration is the method of choice (e.g., infants).
Urinary tract infections (UTI's) are among the most common infections in humans. It has been estimated that approximately 20% of all women will experience at least one UTI, with the incidence increasing with age (Baron & Finegold, Diagnostic Microbiology, 8th ed., p. 254, (C. V. Mosby, 1990)). UTI diagnosis is among the most frequent clinical investigation, with infections of the urinary tract second in frequency only to upper respiratory infections. Indeed, the requests for bacteriuria detection far exceed those for respiratory pathogen detection (Pezzlo, "Detection of Urinary Tract Infections by Rapid Methods," Clin. Microbiol. Rev., 1:268 (1988)). Overall this represents a major cost to laboratories.
The risk of UTI's is significantly increased for patients with indwelling catheters, to the point where it is highly predictable that they will eventually develop at least one UTI. With the ever-increasing number of patients in hospitals and nursing homes with long-term indwelling urinary catheters, this represents a large patient population. Even short-term catheterization presents a significant risk as there is a 20% chance that hospitalized patients with short-term catheters will develop UTI's (Baron and Finegold, supra). Indeed, the National Nosocomial Infections Study (NNIS) conducted by the Centers for Disease Control (CDC) reported that 5% to 6% of all hospitalized patients acquire nosocomial infections. It is estimated that this extends the patient's hospital stay by about 3.2 days and adds approximately $1800 to the direct costs (in 1986 figures). This amount does not take into consideration such factors as physician charges, loss of productivity, and costs associated with deaths (at least 1% of nosocomially infected patients die as a direct result of their nosocomial infection and contribute to the deaths of an additional 2-3% of infected patients).
Most UTI's are acquired by contamination of the urinary tract with the patient's fecal matter. Thus, the members of the Enterobacteriaceae and other organisms present in the patient's gastrointestinal tract are responsible for the majority of UTI's, with E. coli causing the greatest number of infections. The establishment of the gastrointestinal tract as the usual reservoir for UTI's is supported by the observation that the distribution of E. coli serotypes in UTI's corresponds closely with their relative abundance in the affected patient's gut (C. M. Kunin, Detection, Prevention and Management of Urinary Tract Infections, Lea & Febiger ([1979], p. 92). Of particular significance is the association of certain E. coli strains with such serious diseases as hemolytic uremic syndrome (W. R. Gransden et al., "Further evidence associating hemolytic uremic syndrome with infection by verotoxin-producing Escherichia coli O157:H7," J. Infect. Dis., 154:522-534 [1986]), highlighting the importance of E. coli strains in severely debilitating UTI's.
In addition to E. coli, other members of the Enterobacteriaceae have been associated with UTI's. For example, Proteus is frequently isolated in UTI's in boys (Kunin, at pp. 47 and 92). Klebsiella pneumoniae is another important organism in urinary tract infections, as it has been reported to be the second most common pathogen isolated from UTI's (S. Falkow and J. Mekalanos, "The Enteric Bacilli and Vibrios," pp. 561-587 in B. D. Davis et al. (eds.), Microbiology, 4th ed., J. B. Lippincott Co., Philadelphia, 1990]). Indeed, of the Enterobacteriaceae, "80 to 95% of all isolates seen in a general hospital setting will be Escherichia coli, Klebsiella pneumoniae, or Proteus mirabilis." (J. J. Farmer et al., "Biochemical Identification of New Species and Biogroups of Enterobacteriaceae Isolated from Clinical Specimens," J. Clin. Microbiol., 21:46-76 [1985]). Undoubtedly, given the large number of specimens, a major proportion of these isolates are from UTI's. Other species of enteric bacteria are infrequently isolated from UTI's including such noted pathogens as Salmonella and Shigella.
Pseudomonas aeruginosa, an organism that is ubiquitous in the environment can infect almost any tissue or body site, including localized lesions in the urinary tract. UTI's due to P. aeruginosa are more common among the elderly (W. K. Joklik et al., Zinsser Microbiology, Appleton-Century-Crofts, Norwalk, Conn., 1984, p. 631-636). This organism is recognized as being particularly debilitating in patients with underlying disease or immunocompromised conditions.
In addition to the gram-negatives, various gram-positive organisms are commonly associated with UTI's. Enterococcus faecalis is a gram-positive coccus previously included within the genus Streptococcus. Like E. coli (and most species of Enterobacteriaceae), E. faecalis is a member of the normal gastrointestinal flora of humans and may also be found among the normal vaginal flora. Although E. faecalis is associated with various other diseases, UTI's are the most frequent diseases caused by this organism (R. C. Moellering, "The Enterococcus: A versatile pathogen," pp. 3-6, in Challenges in Gram-Positive Injection: A Global Perspective.TM., Healthmark [1988]). Treatment considerations are significant in E. faecalis disease, as this organism is resistant to a large number of antimicrobial agents. For example, E. faecalis is tolerant to a number of antimicrobials that are bactericidal against other bacteria. This high degree of antimicrobial resistance highlights the necessity of identifying this organism from UTI's.
Of the important gram-positive organisms, S. saprophyticus was relatively recently identified as a cause of UTI's (R. H. Latham et al., "Urinary tract infections in young adult women caused by Staphylococcus saprophyticus," J. Amer. Med. Assoc., 250:3063-3066 [1983]; and G. Wallmark et al., "Staphylococcus saprophyticus: A Frequent Came of Urinary Tract Infection Among Female Outpatients," J. Infect. Dis., 138:791-797 [1978]). Prior to the association of this organism with UTI's, it was generally thought that coagulase-negative staphylococci were apathogenic when isolated from the urinary tract (see e.g., B. Hovelius and M ardh, "Staphylococcus Saprophyticus As a Common Cause of Urinary Tract Infections," Rev. Infect. Dis., 6:328-337, 1984). As S. saprophyticus is one of the most common organisms associated with UTI's in young women, the importance of this organism is now recognized. Importantly, not only are these organisms associated with UTI's, they have also been associated with serious infections such as pyelonephritis and sepsis (W. Lee et al., "Pyelonephritis and sepsis due to Staphylococcus saprophyticus," J. Infect. Dis., 155:1079-1080 [1987]). Unlike E. coli and the other enteric organisms, the reservoir for S. saprophyticus remains to be determined.
The organisms discussed above are most commonly associated with ascending infection (A. J. Schaeffer, "Cystitis and Pyelonephritis," pp. 418-435, in Youmans et al., (eds.), The Biologic and Clinical Basis of lnfectious Disease, W. B. Saunders, [1986]). However, organisms may enter the urinary tract by direct extension from the gastrointestinal tract or through hematogenous spread. UTI's may also arise as infections secondary to bacteremia associated with extensive infection at other body sites (Sehaeffer, at pp. 421-423). Hematogenous spread to the kidneys is more common with organisms such as Staphylococcus aureus, Candida sp., and Mycobacterium sp. Thus, organisms may gain access to the structures of the urinary tract through a variety of means, including surgical procedures and catheterization.
The following table lists the organisms commonly associated with hospital and community-acquired UTI's. Notably, a large proportion of these organisms are also residents of the normal gastrointestinal and/or urinary tracts and/or vagina. Table 3 lists the organisms more rarely isolated from UTI's.
TABLE 2 ______________________________________ Organisms Most Commonly Associated With UTI's Acquired In The Community and Hospital Settings Outpatients Hospitalized Patients Initial Recurrent Medical Intensive Cases Cases Wards Care Units Organism (%) (%) (%) (%) ______________________________________ E. coli .gtoreq.90 69 42 24 P. mirabilis 5 8 6 2 Klebsiella- 1 6 13 16 Enterobacter sp. Enterococcus sp. 1 3 15 23 Staphylococcus 1 3 7 5 sp. (coagulase negative) P. aeruginosa 0 &lt;1 6 17 S. marcesens 0 0 1 3 All other organ- 2 11 10 10 isms ______________________________________ *After Clarridge et al., p. 2.
TABLE 3 ______________________________________ Less Common And Unusual Agents Associated With Urinary Tract Infections* ______________________________________ Mycobacterium sp. Leptospira sp. H. influenzae G. vaginalis Acinetobacter sp. Alcaligenes sp. Pseudomonas sp. Citrobacter sp. N. gonorrhoea Salmonella sp. (including S. typhi) Shigella sp. .beta.-Hemolytic Streptococci Anaerobes C. trachomatis T. vaginalis S. haematobium Herpes Virus ______________________________________ *Koneman et al.,; Baron & Finegold; and Power & McCuen.
Although many organisms may be isolated from UTI's, the chances are good that the isolate will belong to one of the organisms listed in Table 2, highlighting the importance of identifying a relatively small number of organisms associated with UTI's.
An additional concern relates to the type of cultures isolated from the urinary tract. Pure cultures are most commonly associated with UTI's in the general population. However, mixed cultures are frequently observed in hospitalized patients.
These mixed infections may present treatment problems, as the therapeutic regimen must be directed to all of the organisms involved. The frequency of mixed cultures is highlighted by a recent study cited by Orenga et al. (supra), in which De Montclos & Carret found that 25% of the urine cultures from hospitalized patients were mixed (De Montclos & Carret, "Optimisation de l'examen cytobacteriolique urinaire," Spectra Biologie, 92:49-53 (1992)). Importantly, mixed infections may also present diagnostic problems, as certain organisms may mask the presence of other species.
Due to the prevalence of UTI's, diagnosis of these infections is a common laboratory procedure. Various methods have been developed for the isolation, identification, and/or detection of the organisms most commonly associated with UTI's. Of these methods, there are two major categories: (1) culture methods, which utilize traditional microbiological culturing techniques to isolate, and then identify microorganisms based on their characteristic biochemical profiles, and for some species, their serological profiles; and (2) non-culture methods, which utilize various enzyme and other systems to detect the presence of infection.
I. Culture Methods for Diagnosis of Urinary Tract Infections
Currently, diagnosis of bacterial UTI's is generally accomplished by means of microbiological culturing and identification of organisms present in urine samples from infected patients. However, a majority of urine specimens submitted to clinical laboratories are negative or have bacterial colony counts below levels considered to be clinically significant (see e.g., Koneman et al.., at 256-257).
Historically, the number of organisms present in a urine sample has been considered to be an important factor in differentiating contaminated samples from those representing true UTI's. Thus, quantitation of the organisms present in a sample is often estimated.
Quantitation may be accomplished by pour plate methods which involve mixing dilutions of a sample with measured volumes of molten agar, pouring the mixture into petri plates, allowing the agar to solidify, incubating for approximately 24 hours, counting the number of colonies present in the plates, and then calculating the number of organisms present in the original sample (Power and McCuen, pp. 48-49; and Clarridge et at., p. 6). While pour plates provide a relatively reliable estimate of the number of organisms present in the sample, the time and manipulations necessary to perform the method make it impractical for use in the clinical setting (Clarridge et al., p. 6).
The method much more commonly used is a streak plate method, in which a calibrated loop designed to deliver a known volume (either 0.01 or 0.001 ml) of urine, is dipped into the sample and the inoculum present in the loop is streaked onto an agar plate (see e.g., E. J. Baron and S. M. Finegold, Diagnostic Microbiology, C. V. Mosby, St. Louis, 1990, pp. 253-262). Following incubation for 18-24 hours, the number of colonies is determined in order to provide an estimate of the number of organisms present in the patient's urine sample.
The commonly used standard is that a count of greater than 10,000 CFU (colony forming units)/ml indicates a UTI. However, the density of pathogens and contaminating organisms in a "positive" specimen may be as low as 100 CFU/ml. Thus, some practitioners identify all bacterial species in numbers greater than 100 CFU/ml, with the exception of normal skin or genital flora (Baron and Finegold, p. 258). A count greater than 1000 has been shown to be significant in males (Clarridge et al., p. 7).
If a specimen contains one or two strains growing in significant numbers, the strains are usually identified and the antimicrobial susceptibility patterns of the strains determined (Baron and Finegold, p. 260). Regardless of the colony count, a pure culture of S. aureus is considered significant (Baron and Finegold, p. 260). Usually, any yeasts isolated are identified to the genus and/or species level, and reported to the physician (Baron and Finegold, p. 260). The following table shows the counts associated with the presence or absence of infection related to the sample volume.
TABLE 4 ______________________________________ Diagnosing Urinary Tract Infections Based On Colony Counts For Three Sample Test Volumes Bacterial Counts (CFU) Sample Size Infection Possible Infection No Infection ______________________________________ Per 1 ml of urine 100,000 1,000 100 Per 10 .mu.l of urine 1,000 10 1 Per 1 .mu.l of urine 100 1 -- ______________________________________
A critical consideration in the cultural diagnosis of UTI's is the choice of media. In order to permit growth of the largest number of species, cultural quantitation methods must be conducted on non-selective, non-inhibitory media (e.g., 5% sheep blood agar, brain heart infusion agar, etc.). Regardless of their purpose as non-selective, selective or differential, most culture media are designed in a manner such that following inoculation of the specimen, the medium is incubated at 35.degree.-37.degree. C., for 18-24 hours or longer, depending upon the organism and medium. Some organisms require different temperatures and/or time of incubation for optimal growth, characteristics which may be helpful in differential diagnosis. Notwithstanding the growth characteristics of the involved microorganism, the treating physician desires as rapid an identification as possible. Thus, primary isolation culture media which permit rapid growth and preliminary presumptive diagnosis of etiological organisms are very desirable. This is of particular importance where immediate treatment is essential.
II. Non-Culture Methods for UTI Diagnosis
Because of the time and manipulation necessary for traditional culture methods, as well as the large number of "negative" specimens, manufacturers and researchers have been very interested in development of rapid urine screening methods. The purposes of these screening methods are: 1) to provide accurate information to the physician in a timely manner, which should correlate with prompt patient care; and 2) to provide rapid elimination of negative specimens, thereby allowing the microbiologist to devote more time to positive specimens, leading to improved cost-effectiveness and efficiency.
Rapid methods described in the literature include microscopic examination (e.g., Gram and acridine orange stains), enzymatic assays (e.g., catalase, glucose oxidase, nitrate reductase, and leukocyte esterase), various endotoxin assays, filtration (e.g., colorimetric), bioluminescent and automated (e.g., photometric detection of growth) procedures. As reviewed by Pezzlo (Pezzlo, "Detection of Urinary Tract Infections by Rapid Methods," Clin. Microbiol. Rev., 1:268, (1988)), these methods have been extensively evaluated. Most compare favorably when a culture method with .gtoreq.10.sup.5 CFU/ml is used as a reference. However, these methods compare less favorably with lower colony counts (Pezzlo, at p. 271). A significant disadvantage of these methods is that they only provide a semi-quantitative estimate of the patient's bacteriuria; they do not provide an indication of the genus or species of the organisms(s) present. This is an important consideration, as the physician needs to know the etiologic organisms in order to provide the patient with the optimum antimicrobial treatment.
From the above, it is obvious that although there are many test systems available, they each have characteristics which preclude their use in certain situations. Many of these tests require expensive reagents, technical time, and laboratory equipment. What is needed is a cost-effective method, at least as sensitive and specific as traditional quantitative culture methods. The method should be rapid, reliable, and should provide at least a presumptive diagnosis to the treating physician in as short a time period as possible.